BACKGROUND: Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells. METHODS: Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure. RESULTS: During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU. CONCLUSIONS: Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.